Figure 7. Retrograde trafficking for the unsaturated C16:1-GM1 species depends on cholesterol, actin and flotillin-1.

A: A431 cells expressing Golgin97-EGFP were depleted of membrane cholesterol with 5 mM MβCD (bottom panels), or not treated (top panels) and loaded with Alexa-labeled C16:1-GM1 followed by treatment with 40 nM CTB.

B: Histogram quantitating the colocalization of C16:1-GM1 with Golgin97 in cholesterol depleted (right columns), or control (left columns) cells treated or not with CTB. Each data point represents a field of view containing 2–3 cells on average.

C: Alexa568 C18:0-GM1 was loaded into A431 cells expressing Rab11-EGFP that were untreated (left panel – M_x = 0.05) or treated with MβCD (middle panel - M_x = 0.13). Panel to right shows cholesterol depleted cells repleted with cholesterol M_x = 0.05. N = 43 cells analyzed per treatment.

D: Histograms quantitating the fraction of C16:1-GM1 colocalizing with the TGN in Golgin97 – expressing A431 cells pre-treated with 20 μM cytochalasin D to depolymerize actin.

E: Fraction of A431 cells with C16:1 or C16:0 Alexa GM1 colocalized in Sec61-EGFP ER/nuclear envelope. Cells transfected with flotillin-1 siRNA and control siRNA were studied and scored by three investigators blinded to treatment groups.

F: Representative immunoblot probed with mouse antibody against flotillin-1 or β-actin in A431 cell lysates.

G & H: same as in panels (E) and (F) using siRNA against flotillin-1 obtained from a different source.

I – K: Histograms quantitating colocalization of the C16:1 Alexa-GM1 isoform with Golgin97 (I), Rab11a (J) and Rab7 (K) in cells transfected with Flotillin1 siRNA (open circles), or untransfected controls (closed circles).

(Bar graphs show mean ± SEM, N = 3 independent scores.)